Scleroderma (systemic sclerosis) is a type of collagen diseases which is a disease whose major symptoms are fibrosis of skin and organs of viscera such as lung, intestine and the like, and disturbances of peripheral circulation. In Japan, it is speculated that there are approximately 10,000 patients, and approximately 500 to 1,000 of new cases are developing every year. Along with the advance in medical science, improvement of the prognoses of various types of collagen diseases have been reported, but the fundamental therapeutic method for scleroderma has not been yet established, and it is a disease whose prognosis is so bad that 10-year survival rate is less than 70%. Symptoms in patients suffering from scleroderma vary and have a wide range from the patients with only extremely light disturbances of circulation who require no treatment at all to the patients who die of respiratory failure, renal failure, cardiac failure or the like within a short period. Therefore, it is extremely important to predict what kind of organ failure will progress in the future of a patient diagnosed as having scleroderma.
In the sera from the patients suffering from scleroderma, autoantibodies (antinuclear antibodies) against nuclear protein which have important biological activities such as topoisomerase I, centromere and the like, are detected with high frequency. As these autoantibodies are specific to scleroderma, they are used in its diagnosis. Further, as anti-topoisomerase I antibodies are associated with diffuse cutaneous scleroderma accompanying pulmonary fibrosis where its range of skin sclerosis is wide, while anti-centromea antibodies are associated with limited cutaneous scleroderma where visceral lesions are few and skin sclerosis is localized in fingers, detecting these antinuclear antibodies are also useful in classifying clinical entities and predicting future organ failures (Arthritis Rheum, 37:75-83, 1994). The antinuclear antibodies, which are specific to scleroderma and useful in diagnosing and classifying clinical entities, are shown in table 1, and among them whose measuring kits are released, and it is measurable in general practice, are only 3 kinds, i.e., anti-topoisomerase I antibodies, anti-centromere antibodies and anti-U1RNP antibodies.
TABLE 1Range ofAntinuclearPositiveskinVisceralantibodyfrequencysclerosislesionAnti-topoisomerase25%DiffusePulmonaryI antibodiesfibrosis,(Anti-Scl-70Skinantibody)ulcerAnti-centromere20%LimitedRareantibodiescardiac,renal andpulmonary,failures,PrimarybiliarycirrhosisAnti-U1RNP20%LimitedMyositis,antibodiesPulmonaryhypertensionAnti-RNA 5%DiffuseSclerodermpolymerasea renalantibodiescrisis,MyocardialfailureAnti-U3RNP 3%DiffuseFewantibodyvisceralfailuresAnti-Th/To 2%LimitedFewantibodyvisceralfailuresAnti-PM-Scl<1%LimitedMyositisantibodiesAnti-Ku antibodies 2%LimitedMyositis
Anti-RNAP antibodies are antibodies found in the sera from patients suffering from scleroderma were identified as antinuclear antibodies recognizing several RNA polymerases (RNAPs) simultaneously for the first time in 1993 by the present inventors (J Clin Invest 91:1399-404, 1993). RNAP is an enzyme catalyzing the transcription of RNA, and RNAP I, RNAP II and RNAP III transcribe ribosomal RNA, messenger RNA and transfer RNA, respectively. Antibodies that simultaneously recognizing RNAP I and RNAP III were detected in the sera from patients suffering from scleroderma, and a part of which also reacts to RNAP II. Anti-RNAP antibodies have extremely high specificity to scleroderma, which has not been reported heretofore except in patients suffering from scleroderma. Anti-RNAP antibodies are detected with high frequency in diffuse cutaneous scleroderma where skin sclerosis progresses rapidly, and they supervene scleroderma renal crisis at a high rate and associate with clinical entities whose prognoses are extremely bad. Before 1980s when there was no therapeutic method for scleroderma renal crisis, the 5-year survival rate was only 30%. Many of fatal cases die of complication called scleroderma renal crisis where blood pressure rises acute, and renal failures are accompanied. Since the beginning of 1990s, a specific medicine against scleroderma renal crisis (ACE inhibitor) was found, and it was shown that administrating them in the early phase of sideration of scleroderma renal crisis allowed the patients to survive without leaving a sequela. However, although an ACE inhibitor is administered after the symptoms of scleroderma, renal crisis progress and renal function changed for the worse. Effectiveness of the ACE inhibitor is not expected. Even including fatal cases, although the patients can recover, dialysis is necessary for many of them. Therefore, the patients detected anti-RNAP antibodies related to scleroderma renal crisis recommended ensuring the self-blood pressure measurement for early detection of scleroderma renal crisis. Consequently, anti-RNAP antibodies are extremely useful antinuclear antibodies in diagnosing and classifying clinical entities of scleroderma, and predicting organ failure of scleroderma, in particular scleroderma renal crisis. Further, it is thought that examining the presence of anti-RNAP antibodies in diagnosing brings about the early detection of scleroderma renal crisis, decrease of the fatal cases by early therapy and improvement of life prognoses of scleroderma.
It has been reported by the present inventors that positive frequency of anti-RNAP antibodies in Japanese patients suffering from scleroderma is as low as about 5%, while in Caucasian patients in Europe and the United States, the rate is as high as 20-30%, whose frequency is higher than that of anti-topoisomerase I antibodies, anti-centromere antibodies and anti-U1RNP antibodies, and that anti-RNAP antibodies are the highest detected antinuclear antibodies in scleroderma in Caucasian in Europe and the United States (Arthritis Rheum 37, 902-6, 1994). Then, these results have been confirmed by the supplementary examinations in several facilities in Europe and the United States (Ann Intern Med, 119, 1005-13, 1993, Clin Exp Immunol, 105, 468-74, 1996).
Although anti-RNAP antibodies are clinically very useful antinuclear antibodies, a cumbersome immunoprecipitation which uses large amount of isotopes and cultured cells is the only method of detecting currently, therefore the present situation is that detecting anti-RNAP antibodies in a general clinical laboratory is thought to be impossible, and the detection can be performed only in specialized laboratories. Consequently, the development of a convenient method of detecting anti-RNAP antibodies is thought to be an urgent task. However, anti-RNAP antibodies cannot be detected by double immunodiffusion and immunoblotting by using crude antigens which are commonly performed for detecting antinuclear antibodies. The both of RNAP I and RNAP III are giant complexes comprising 10 or more subunits, and the number of molecules of respective subunits per cell are extremely few, can be exemplified as its reasons. In order to solve this point, identifying antigenic sites (epitopes) which are commonly recognized by anti-RNAP antibodies in the sera from patients suffering from scleroderma is essential. Development of the method of detecting including enzyme linked immunoassay (ELISA) is thought to be possible by expressing the sites in a large amount as recombinant proteins, when epitope regions on the RNAP I and RNAP III molecules become clear. An object of the present invention is to provide a convenient method of detecting anti-RNAP antibodies, which is extremely useful in diagnosing and classifying clinical entities of scleroderma, and predicting organ failure, in particular scleroderma renal crisis, and which has extremely high specificity to scleroderma, as they are detected with high frequency in the patients suffering from diffuse cutaneous scleroderma where skin sclerosis progresses rapidly, and they supervene scleroderma renal crisis at a high rate and related to clinical entities whose prognoses are extremely bad.
A basic study by the present inventors resulted in the elucidation that anti-RNAP antibodies in the sera from patients suffering from scleroderma recognize 2 subunits of 155-kDa and 62-kDa (RPC155, RPC62) of RNAP III with high frequency (Arthritis Rheum, 42, 275-84, 1999). In addition, cDNA sequence of human RPC155 was reported by Setareh and Hernandez in 1997 (Genome Res, 7, 1006-19, 1997), and it is registered to database of NCBI (accession No. AF021351). Then, in order to identify the epitopes existing on RPC155 and RPC62 in more detail, the full-length protein of RPC62 and a partial fragment of RPC155 were expressed as recombinant proteins in Escherichia coli and the reactivities to sera positive and negative to the anti-RNAP antibody from patients suffering from scleroderma were examined by immunoblotting. First, study using the recombinant proteins of RPC155-A (Seq. ID No. 8), RPC155-B2 (Seq. ID No. 10), RPC155-C (Seq. ID No. 12), RPC155-D (Seq. ID No.14) and RPC62 (full-length) shown in FIG. 1, led to the result shown in Table. 2. Each recombinant protein was recognized by sera positive to anti-RNAP antibody, while all of 11 sera positive to anti-RNAP antibodies reacted to RPC155-C.
TABLE 2SclerodermaSclerodermaRecombinantAnti-RNAPAnti-RNAPNormalRNAP IIIAntibody(+)Antibody(−)PersonFragment(n = 11)(n = 10)(n = 6)RPC155-A2(18%)1(10%)0RPC155-B21(9%) 00RPC155-C11(100%)00RPC155-D3(27%)00RPC626(55%)00
There, in order to examine epitopes contained in RPC155-C in more detail, 5 recombinant fragments of C1-C5 shortened from N- and C-terminals of RPC155-C were newly generated. When the reactivities to 11 sera that were positive to anti-RNAP antibodies were studied by immunoblotting, all of the sera recognized C3 and C4, while they did not react to C1, C2 and C5. Further, 7 recombinants of C-a to C-g encoding the common site of C3 and C4 were generated to examine the reactivities to sera positive to anti-RNAP antibody similarly. All of the sera reacted to C-c and C-g, while they did not recognize other recombinant fragments. Consequently, it was revealed that there are epitopes recognized commonly by anti-RNAP antibodies in the sera from patients suffering from scleroderma, at No. 891-1020 amino acid residues of RPC155 encoded by C-c. However, as the reactivity to C-c was weaker than that to C-g in certain sera, it was predicted that an amino acid site contained additionally in C-g was also necessary for strong binding to antibodies in the sera from patients. Therefore, it was revealed that C-g is more suitable than C-c for detecting anti-RNAP antibodies.
In order to confirm that the reactivity to C-g is specific to anti-RNAP antibodies in the sera from patients suffering from scleroderma, the study by immunoblotting with more examples was performed. As a result, the reactivity to C-g was detected in all of the 16 positive cases of scleroderma to anti-RNAP antibody, while it was not detected in 55 negative cases to anti-RNAP antibody scleroderma and 26 cases of normal person. Consequently, it was thought that C-g comprises epitopes recognized commonly by anti-RNAP antibodies in the sera from patients suffering from scleroderma, and recombinant proteins expressing the same sites are useful as antigens in methods of detecting anti-RNAP antibodies. Then, after C-g was expressed in Escherichia coli, it was purified by affinity column, and used as an antigen for ELISA. As shown in FIG. 2, reactivity to C-g in sera negative to anti-RNAP antibody scleroderma, the sera from patients suffering from systemic lupus erythematosus and sera from normal person used as controls tested by conventional immunoprecipitation were at a low level. When 4.15 units represented by the average of data+5×standard deviation from normal person was made to be cutoff, positive frequency of anti-C-g antibodies showed 100% in examples positive to anti-RNAP antibody and showed 0% in other examples, which revealed that ELISA by using C-g is an extremely superior method of detecting anti-RNAP antibodies where both sensitivity and specificity were 100%. The present invention has been completed based on these knowledges.